Extended-spectrum beta-lactamases found in Escherichia coli isolates obtained from blood cultures and corresponding stool specimen

With extended-spectrum β-lactamases (ESBLs) and CTX-M enzymes being on the rise, antimicrobial treatment of enterobacterial infections is becoming more and more challenging. Our study aimed at a molecular characterization of phenotypically ESBL-positive E. coli strains obtained from blood cultures of patients of the University Hospital of Leipzig (UKL), Germany. The presence of CMY-2, CTX-M-14 and CTX-M-15 was investigated using Streck ARM-D Kit (Streck, USA). Real-time amplifications were performed by QIAGEN Rotor-Gene Q MDx Thermocycler (QIAGEN, Thermo Fisher Scientific, USA). Antibiograms as well as epidemiological data were evaluated. Among 117 cases, 74.4% of the isolates showed a resistance to ciprofloxacin, piperacillin and ceftazidime or cefotaxime while being susceptible to imipenem/meropenem. The proportion of ciprofloxacin resistance was significantly higher than the proportion of ciprofloxacin susceptibility. At least one of the investigated genes was detected in 93.1% of the blood culture E. coli isolates: CTX-M-15 (66.7%), CTX-M-14 (25.6%) or the plasmid-mediated ampC gene CMY-2 (3.4%). 2.6% were tested positive for two resistance genes. 94 of the corresponding stool specimens tested positive for ESBL producing E. coli (94/112, 83.9%). 79 (79/94, 84%) E. coli strains found in the stool samples matched with the respective patient’s blood culture isolate phenotypically (MALDI-TOF, antibiogram). The distribution of resistance genes was in accordance with recent studies in Germany as well as worldwide. This study provides indications of an endogenous focus of infection and emphasize the importance of screening programs for high-risk patients.

www.nature.com/scientificreports/ In this context and considering the rising antimicrobial resistance of E. coli due to extended-spectrum β-lactamases, this study aimed at the molecular characterization of ESBLs detected in E. coli strains obtained from blood cultures of patients of the University Hospital of Leipzig (UKL), Germany. Furthermore, our study provides epidemiological data and resistance patterns of the isolated strains. In order to find indications of an endogenous focus of infection for the E. coli bacteremia, the laboratory database was searched for corresponding stool samples with evidence of ESBL producing E. coli for each patient. Whenever available, resistance patterns from both strains (blood culture and stool sample) of the individual patients were compared.

Materials and methods
E. coli strains. The E. coli strains investigated with this study were all collected between 2015 and 2018 from patients of the UKL. They were either blood culture isolates or strains that were recovered during a continuous risk-adapted screening of stool samples for multiple drug resistant strains.
As part of this screening, stool samples and rectal swabs are examined for the presence of carbapenemase-and β-lactamase-producing bacteria employing the chromogenic media CHROMagar ESBL and CHROMagarTM ESBL (Mast Diagnostica, Germany) as initial test procedure. The stool and rectal samples were collected as part of this UKL screening program and were neither collected by any of the authors nor for the purposes of this study in the first place. Therefore, there were no human participants involved in this study. All strains recovered were identified with MALDI-TOF (BioMerieux, France). Altogether, 117 strains from blood cultures (ECB) with phenotypic evidence of ESBL production and 112 strains recovered from screening (ECS) were considered for further analysis. For 94 of the bacteremic patients ESBL positive stool screening cultures were available. Those as well as all blood culture isolates were further analyzed with this study. Susceptibility test. All E. coli isolates were submitted to broth microdilution performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Minimum inhibitory concentrations (MICs) for the following antibiotics were determined: ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefotaxime, cefuroxime, aztreonam, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, colistin, fosfomycin, trimethoprim/sulfamethoxazole and tigecycline. E. coli ATCC 25922 was used as quality control strain.
ESBL detection. Phenotypic testing for ESBL production was performed for all isolates with a MIC of ≥ 1 mg/l for either cefotaxime, ceftazidime or aztreonam. For this purpose, a gradient test system using cefotaxime/clavulanic acid and ceftazidime/clavulanic acid test strips (Etest®, bioMérieux, France) was employed. According to the recommendations of the manufacturer, individual bacterial colonies were transferred to 0.5% NaCl solution until a MC Farland standard of 0.5 was obtained. The suspension was then streaked onto Müller-Hinton agar, the strips were applied, and the tests incubated for 18 to 24 h at 37 °C. All isolates positive in the gradient test were then analyzed for the presence of extended-spectrum beta-lactamase genes CTX-M-14 and CTX-M-15 and the plasmid-mediated ampC gene CMY-2. Data collection and evaluation. Antibiograms and epidemiological data were collected by laboratory IT Hybase (epiNET AG, Germany) and LabCenter (i-solutions health GmbH, Germany). HyBase® as well as Microsoft Excel were used for the following evaluations.

Results
In total, 130 ESBL producing E. coli isolates were preserved and included in the further investigation. Since 13 isolates turned out to be duplicates of the same patient, those were not included in any statistical evaluation leaving 117 isolates for further analysis.

ECB-data.
During the time period of 2015 to 2018, 159,074 blood cultures were submitted for microbiological evaluation for patients of the UKL. Out of these, 26,427 yielded bacterial growths (positivity rate: 16.6%). Since multiple cultures were submitted for individual patients with positive culture (n = 9910), a corresponding diagnosis of bacteremia was made in 6.2% of all cases. Altogether, in 1020 cases a bacteremia with E. coli was documented (10.3%) including 153 cases where ESBL producing strains were detected (15%). Thus, out of 9910 patients suffering from bacteremia, ESBL producing E. coli strains were documented in 1.5% of the cases. In total, 130 ESBL producing E. coli isolates were collected and included in the investigations. Among these, 13 isolates were duplicates of individual patients, so they were eliminated from any statistical evaluation leaving 117 isolates for further interpretation.

ECS-data.
Of the 117 bacteremic patients included in this study, 112 had submitted stool samples prior to the time of bacteremia. 94 of these specimens tested positive for ESBL producing E. coli (94/112, 83.9%). Retrospectively, a total of 79 (79/94, 84%) E. coli strains found in the stool samples matched with the respective patient's blood culture isolate phenotypically (MALDI-TOF, antibiogram). The comparison between the stool samples and blood cultures that tested positive for ESBL is shown in Table 2. Considering the ciprofloxacin resistance, 55.4% (62/112) of the patients were colonized with an ESBL positive and ciprofloxacin resistant strain. 15 (15/94, 16%) of the isolates showed no phenotypic match of blood culture isolate and respective stool sample. Table 3 shows the epidemiology of multi-resistant E. coli during the investigation period. Between 2015 and 2018 there were 12,346 cases of infection by invasive E. coli, id est isolations of E. coli excluding rectal swabs and stool samples. Overall, 13.1% (1614/12,346) were caused by ESBL-producing isolates, while 8.0% (988/12,346) of these were caused by ESBL-producing strains with additional ciprofloxacin resistance; within ESBL-positive isolates, the proportion of ciprofloxacin resistance (61.2%, 988/1614) was higher than the proportion of ciprofloxacin susceptibility significantly. Further, 1020 cases of bloodstream infections by E. coli were detected during the investigation period. Among the 117 cases included in this study, 74.4% of the isolates were showing a resistance to ciprofloxacin, piperacillin as well as ceftazidime or cefotaxime while being susceptible to imipenem/ meropenem.   15 .

Infection due to ESBL-E. coli.
Since an extensive screening program for multidrug resistant microorganisms is in place in our hospital, we were also able to address the fecal carriage of ESBL producing E. coli strains of the patients with bloodstream infections of our study. Interestingly, of the 117 bacteremic patients included in this study, 112 had submitted stool samples prior to time of bacteremia and 94 of these specimens yielded ESBL producing E. coli (94/112, 83.9%). 79 of the E. coli strains found in the stool samples matched with the respective patient's blood culture isolate phenotypically, suggesting a high incidence of endogenous infections. Reddy et al. reported earlier on an infection control program addressing ESBL producing Enterobacteriales-they found that out of 413 colonized patients 8.5% developed a subsequent bloodstream infection, amounting to 34.3% of all bloodstream infections by ESBL producing Enterobacteriales 16 . Our data emphasize once more the value of risk adapted screening and-in case of an infection-the necessity to select an empiric therapy accordingly. Moreover, the epidemiology of the underlying ESBL genes should be surveyed on a regular basis and analyzed together with the respective susceptibility data.
With this study E. coli and three genes were addressed, only. This is an obvious shortcoming and we cannot rule out that other ESBL genes were also present in our isolates. However, in 109 out of the 117 isolates (93.1%), there was at least one of the investigated genes detected and the results are in line with the epidemiology reported globally.

Conclusion
The molecular investigation of ESBL producing E. coli collected from blood cultures from patients at the University Hospital of Leipzig, Germany, between 2015 and 2018 showed a distribution of resistance genes that was in accordance with recent studies in Germany as well as worldwide with CTX-M-15 being the most prevalent genotype in our study.
The results of our study provide possible indications of an endogenous focus of infection for an E. coli bacteremia and, therefore, emphasize the importance of screening programs for high-risk patients.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.